Phase II project, submitted by InSilixa, Inc., a Silicon Valley start-up company, proposes to leverage Phase I accomplishments by developing a complementary metal-oxide- semiconductor (CMOS) biochip platform for the diagnosis of infectious diseases. This project addresses the growing global threat of antibiotic resistant bacteria whose emergence and dissemination now complicates antibiotic selection decisions, negatively affects prognosis, increases health care costs, and fuels increasing levels of resistance. To tackle this crisis, InSilixa will develop a highly multiplex platform that can simultaneously, rapidly and inexpensively detect an infectious agent in a clinical sample and identify the mutations that confer drug resistance. Specifically, the work proposed in this application will entail the design, implementation, and experimental validation of a nucleic acid amplification testing (NAAT) method that uses the proprietary HYDRA CMOS biochip detection platform. Aim 1 will develop a single-chamber, closed-tube, and highly multiplexed NAAT technology that quantifies, in real- time and in parallel, 10's of multiplex PCR reactions and identifies and characterizes the sequences of the generated amplicons by performing 100's of high-resolution melt curve analysis (MCA) experiments. Aim 2 will further develop the CMOS biochip detection platform (HYDRA-4K). And Aim 3 will develop a comprehensive NAAT assay based on combining the above technologies for detecting antimicrobial-resistance in M. tuberculosis. Though the initial clinical focus is tuberculosis, the design, development and manufacturing know-how gained during the course of this project will enable all other applications (and products) for the HYDRA platform. Included amongst these are attractive commercial opportunities including respiratory tract infections, drug resistant hospital acquired infections, outpatient urinary tract and pharyngeal infections and the identification, quantificatin and drug susceptibility genotyping of HIV in the blood of HIV/AIDS patients. The successful completion of this Phase II project will result in a fully characterized, working prototype of the InSilixa CMOS biochip platform that can simultaneously identify an infectious agent and the mutations that confer resistance to a large panel of antibiotics.